Circulating tumour cell (CTC) refers to collectively all types of tumour cells occurring in peripheral blood, which was detached from a solid tumour lesion (primary lesion or metastatic lesion) spontaneously or due to medical operations. After entering into peripheral blood, most CTCs ended in apoptosis or being devoured, while a few may escape and anchor to develop into a metastatic lesion, resulting in increased mortality risk of malignant tumour patients.
Research in recent years has revealed that tumour cells, during the process of entering peripheral blood (PB) circulation, may go through epithelial-mesenchymal transition (EMT). Tumour cells gone through epithelial-mesenchymal transition (EMT) have increased migration and invasion abilities. Some researchers detected the expression of CTC mesenchymal marker vimentin and epithelial marker Keratin mRNAs in metastatic NSCLC patients using immunofluorescent staining, wherein results indicating that strong co-expression of both markers were observed in nearly all CTCs. No CTCs that only expressed keratin mRNA were observed, while a few CTCs that only expressing vimentin mRNA were found in 3 patients. This study confirmed for the first time the existence of CTCs with mixed epithelial/mesenchymal phenotypes. The phenotype of NSCLC primary tumour is keratin mRNA positive and vimentin mRNA negative. In addition, other researchers have conducted assays for determining three EMT marker mRNAs (TWIST1, Akt2, PI3Kα) of CTCs in breast cancer patients using AdnaTest method, and found that 29% patients appeared positive for at least one of these markers. By analyzing the single cell transcriptional profiles of CTCs in breast cancer patients, researchers found that, despite of gene expression variations among CTCs, genes that involved in EMT, including TGF-β1, vimentin and CXCR4, are generally highly expressed in CTCs. Other studies also confirmed the highly frequent occurrence of co-expression of epithelial markers (EpCAM, cytokeratins, E-cadherin), mesenchymal markers (vimentin, N-cadherin, O-cadherin), and stem cell marker (CD133) in CTCs of tumour patients. In yet another study, researchers performed assays to determine expression of EMT-relevant transcription factor mRNAs (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2) in breast cancer CTCs and analyzed potential influences of presence or absence of neoadjuvant chemotherapy on expression of such transcription factors in CTCs. They found that neoadjuvant chemotherapy did not eliminate these CTCs in which EMT had occurred. It was proposed that EMT probably provided the CTCs with tolerance to chemotherapy by endowing them with stem cell characteristics. Therefore, CTCs typing and identification may have great significance in guiding development of molecular targeted drugs and clinical individualized treatment.
Currently, most detection methods for CTCs use epithelial markers (such as epithelial cell adhesion molecule, EPCAM) as target spots, then capture CTCs with corresponding antibodies, and use CKs expressed on cells as a primary diagnostic indicator. Such methods involve EPCAM and CKs that are both specific for epithelial cells. A representative detection method in this regard is CellSearch system, which is the only one that is approved by USFDA for clinical application. False positive or false-negative detection results may occur due to the possible presence of a number of non-neoplastic epithelial cells in peripheral blood, potential contamination in blood samples by normal epithelial cells during blood sampling, and that some CTCs cannot be detected due to loss of epithelial antigens during EMT. Additionally, immunomagnetic separation (MACS) technique in combination with reverse transcriptase-polymerase chain reaction (RT-PCR) is also a commonly used CTC separation and identification technique. However, drawbacks exist as RT-PCR process has high requirements for environments and operation, mRNA is prone to degradation which disables CTC cell typing, and the like. Therefore, a technique for accurate detection, identification, and precise typing of CTCs is in urgent need.